Whether you are a biologist, biochemist, physician or pharmaceutical developer, chances are that you have wanted to know the chemical composition of a sample. One of the most popular ways to make this determination is reversed-phase liquid chromatography, which separates compounds into bands for identification. However, chromatography bands can become broad and even smear together, which hinders interpretation.

Reversed-phase liquid chromatography is necessary not only for determining the composition of samples but also for separating molecules. For example, the pharmaceutical industry routinely uses chromatography to isolate drugs from unwanted products. Just as band broadening can prevent a complete understanding of sample composition, it also can prevent complete separations.

Using confocal ratiometric imaging and single-molecule spectroscopy of chromatography columns, Zhenming Zhong and M. Lei Geng from the University of Iowa in Iowa City recently discovered a source of band broadening. Geng said that they chose the combination of confocal imaging and single-molecule spectroscopy because it enables direct observation of the separation process in real time and with high spatial resolution.

In chromatography, a mobile phase pushes the sample through a stationary phase designed to interact with molecules to various degrees. Because of these interactions, the stationary phase retains some compounds for a longer time than others, so compounds become separated from each other.

The researchers studied a chromatography column that uses porous silica beads as the stationary phase. The surface of the pores is covered with a single layer of hydrocarbon chains containing 18 carbon atoms per chain (C18-silica beads). “C18-silica is the most common stationary phase used in industry and research labs today,” Geng said. In particular, the researchers used Luna beads from Phenomenex Inc. of Torrance, Calif., because they have shown outstanding performance in separations.

Geng also said that they believe that the results in this study are common to other brands of C18-silica columns; they are investigating the polarity of other brands as well. Because chromatography separates compounds based on their affinities to the low-polarity stationary phase, the investigators used a polarity-sensitive fluorophore, Nile red, to probe the phase.

‘Supersensitive’ setup

Geng said that they needed to use a “supersensitive” setup to enable single-molecule spectroscopy (Figure 1). They employed a Nikon inverted microscope and a Melles Griot air-cooled argon-ion laser at 514.5 nm for fluorescence excitation. “The TEM00 single-mode laser is focused down to the diffraction limit to achieve the highest resolution,” Geng said. They used an X-Y piezoflexure stage to move the chromatography column, and they made Z-stacks with a piezoelectric objective stepper, both from Physik Instrumente LP of Karlsruhe, Germany. According to Geng, the high resolution of the stage movements ensured that the spatial resolution of the images was determined by optics and not by stage movement. The researchers coordinated stage movement using programs written in National Instruments’ LabView.

Figure 1. The sensitivity of the researchers’ setup enabled them to do single-molecule spectroscopy in two spectral channels on beads within chromatography columns. Images reprinted with permission of Analytical Chemistry. (MCS = multichannel scaler, APD = avalanche photodiode).They placed the chromatography column on the stage and surrounded it with refractive-index matching oil. Without the oil, there would be an interface between the air and the column that would cause light to refract and reflect according to Fresnel’s law, resulting in unwanted position and intensity distortions to the image. The 100× oil-immersion objective had a numerical aperture of 1.45 to maximize light-collection efficiency.

Detection of the fluorescence signal was accomplished by two PerkinElmer Optoelectronics avalanche photodiodes. A multichannel scaler for each avalanche photodiode ran on a personal computer and counted the photons. Geng said that they chose multichannel scalers because they have a short dead time — the time in which photons are lost during photon-count information transfer and storage. After collecting the data as a line graph of intensity versus time, they used MathWorks Inc.’s MatLab to convert the data into ratiometric images. They used the publicly available ImageJ to look at specific beads in the chromatography column.

The researchers reported their findings in the Sept. 1 issue of Analytical Chemistry. Using confocal ratiometric imaging, they discovered that the overall polarity of each bead can differ significantly from that of neighboring beads (Figure 2). Geng said that this was their most important finding because the difference in polarity among the beads will contribute to band broadening and, thus, decrease the resolution of separations.

Figure 2. The researchers observed dramatic differences in overall polarities among beads, such as in beads A through F, shown here. The coloration of the beads is the result of the polarity-sensitive Nile red fluorophore.Polarity differences

It was possible that the overall polarity differences could be the result of exposed silanols on the surface of the pores of the beads, especially because surface silanols are known to affect separations by causing peak tailing, broadening of the trailing end of the band and of the corresponding spectral peak. Again using ratiometric imaging, the researchers determined that the polarity throughout each bead was uniform. Single-molecule spectroscopy observations combined with ratiometric imaging led them to conclude that large clusters of exposed silanols did not exist in silica beads, thus excluding silanols as the cause of the polarity differences. Geng said that these findings mean that the cause of the overall polarity differences remains to be determined.

The scientists hypothesized that the differences in the size of pores within the beads could affect the packing density and structure of the hydrocarbon layer and cause the overall polarity differences. According to Geng, the arrangement and structure of the pores within the beads also could be a factor.

He said that they plan to investigate the reason for differences in polarities. He believes that one way that the chromatography columns can be made to have more uniform polarities is first by separating the beads based on polarities and next by packing the beads with similar polarities into small-scale chromatography columns, such as those used for capillary or microchip high-performance liquid chromatography. He said that, although this method may be time-consuming, flow cytometry could speed it up. He expects that future improvements in stationary phase preparation will generate columns with uniform polarity.